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Glycosylation modification method of huntingtin protein

A technology of huntingtin protein and modification method, which is applied in the field of chemical modification of protein, and can solve problems such as poor stability of huntingtin protein

Active Publication Date: 2015-09-30
浙江璞题生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Unmodified huntingtin protein shows poor stability, etc. At present, there are no research institutes or institutions in China to chemically modify huntingtin protein

Method used

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  • Glycosylation modification method of huntingtin protein
  • Glycosylation modification method of huntingtin protein
  • Glycosylation modification method of huntingtin protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Obtaining recombinant protein Htt1-90

[0028] 1) Test material

[0029] (1) Vector and strain

[0030] Plasmid pcDNA3.1 / myc-His was donated by CHDI; expression vector pTWIN1 was purchased from NEB Company; plasmid pMD18-T and Escherichia coli JM109, DH5α were purchased from Takara Biotechnology Co., Ltd.

[0031] (2) Primers

[0032] P1: 5-CCG GAATTC CTGCCGTGCC-3 (EcoR I)

[0033] P2: 5-AAAA CTGCAG ACAGCCGGGC-3 (Pst I)

[0034] Synthesized by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment approach

[0036] 1. Obtaining the Huntingtin Gene

[0037]Using the plasmid pcDNA3.1 / myc-His as a template, design and synthesize upstream and downstream primers P1 and P2 containing EcoR I and Pst I restriction enzyme sites to amplify the htt gene fragment by PCR, and detect the PCR product by agarose gel electrophoresis Recovered, purified and ligated with vector pMD18-T, transformed into Escherichia coli JM109, and screened for ampicillin resistance to obtain recombinant plasmid pMD18T-htt, which was sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. The verified recombinant plasmid was digested with EcoR I and Pst I, and the digested product was purified and stored in a refrigerator at 4°C.

[0038] 2. Construction of recombinant expression plasmid pTWIN1-htt

[0039] The prokaryotic expression vector pTWIN1 was double-digested with restriction endonucleases EcoR I and Pst I, and after purification, the linear pTWIN1 plasmid fragment and the htt gene fragment were lig...

Embodiment 2

[0043] Embodiment 2: Purification of recombinant protein Htt(1-90)

[0044] The CBD component of the Htt1-90-intein-CBD fusion protein can be specifically combined with chitin resin to facilitate the removal of foreign proteins, and then the intein component catalyzes the fusion protein to break between htt1-90 and intein under certain conditions. The specific operation is as follows: the supernatant is loaded on a 2ml chitin gravity column. The column was first equilibrated with solution A (Na-HEPES (pH8.0) 20mmol / L, NaCl 500mmol / L, EDTA 1mmol / L), and the supernatant was loaded and then eluted with solution A. Then the column was immersed in solution B (Na-HEPES (pH8.0) 20mmol / L, NaCl 500mmol / L, EDTA 1mmol / L, DTT 50mmol / L) at 40C for 16h. Solution C (Na-HEPES (pH8.0) 20mmol / L, NaCl 500mmol / L) eluted protein and collected every 1ml. The samples collected in each step were analyzed by SDS-PAGE, and the purity of the identified protein was 90%.

[0045] A solution containing ...

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Abstract

The invention discloses a glycosylation modification method of hungtintin. The glycosylation modification method comprises the following steps: (1) obtaining a recombinant protein Htt(1-90), to be specific, a. obtaining a hungtintin gene, b. constructing a recombinant expression plasmid pTWIN1-htt, c. constructing a recombinant escherichia coli strain DH5 alpha-htt for expressing the hungtintin, and d. expressing the recombinant protein; (2) purifying the recombinant protein Htt(1-90); (3) obtaining a glycosylation modified Htt(Cys-K92-K158) polypeptide by using an SPPS (stable protein plasma solution) method; (4) purifying the Htt(Cys-K92-K158) polypeptide; (5) coupling the Htt(Cys-K92-K158) polypeptide with the recombinant protein Htt(1-90) so as to obtain a glycosylation modified target protein Htt(1-158); (6) purifying the glycosylation modified target protein Htt(1-158). The glycosylation modification on the hungtintin shows that the glycosylated hungtintin has relatively strong stability; the influence of the temperature and the time on absorption peaks of the hungtintin shows that the hungtintin subjected to glycosylation modification is relatively stable.

Description

technical field [0001] The invention relates to a chemical modification of protein, in particular to a method for glycosylation modification of huntingtin protein. Background technique [0002] Protein modification is a complex process. There are many types of modification in eukaryotes. The common ones are glycosylation, acetylation, ubiquitination, phosphorylation and SUMOylation. Protein modification can change the activity, location or function of proteins. Protein glycosylation has an important impact on the function and stability of proteins. First of all, many misfolded proteins have a certain impact on cell activity and tissue metabolism, which in turn affects the function of organs. The connection forms of glycosylated proteins mainly include N , O connection two forms. The change of protein structure has a certain influence on circular dichroism absorption and resistance to hydrolytic enzymes; secondly, some glycosylated proteins are conducive to the correct foldi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K1/107C12N15/70
CPCC07K14/47C12N15/70
Inventor 王喆明
Owner 浙江璞题生物科技有限公司
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